Project Summary/Abstract: The mitochondrion is a unique organelle that serves as the main site of adenosine triphosphate (ATP) generation needed for energy in the cell. However, mitochondria also play essential roles in cell death through apoptosis and necrosis, as well as a variety of crucial functions related to stress regulation, autophagy, lipid synthesis, and calcium storage. There is a growing appreciation that mitochondrial function is regulated by the dynamics of its membrane fusion and fission; longer, fused mitochondria are optimal for ATP generation, whereas fission of mitochondria facilitates mitophagy and cell division, events that have a direct bearing on cancer tumorigenesis. Despite the significance that mitochondria play in regards to cancer cells, the intricate regulation of mitochondrial function is only partially understood. Only now, have researchers turned to address the upstream machinery that regulates mitochondrial function through fusion and fission. The proposed research strategy will open up new avenues for translational and future clinical studies on the modulation of mitochondrial function, allowing for novel therapeutics for cancer patients. The first aim of this proposal, to elucidate the mechanism how EHD1 and Rabankyrin-5 regulate mitochondrial fission, will continue to build upon recent findings that indicate significant upstream regulation of mitochondrial fission by endocytic regulatory proteins. This portion of the proposal will characterize the mechanism by which EHD1, an endocytic regulatory protein, and its interaction partner, Rabankyrin-5, regulate VPS35, an endocytic protein that modulates mitochondrial fission. The mechanism by which EHD1 and Rabankyrin-5 regulate VPS35 will be elucidated by studying the physical and functional relationships between EHD1 and VPS35 by various techniques, including CRISPR/Cas9 gene-edited cells, targeted amino acid substitutions in the EHD1 domains, advanced imaging techniques, purified proteins, and GST pull-downs. Characterization of the interaction between EHD1 and VPS35 will further add to the complex upstream regulation of mitochondria fission, which is essential for the proper mitochondrial function that is lacking in cancer cells. The second aim of this proposal, to explore a novel role for endocytic proteins in the trafficking of BCL-2 family members and the regulation of apoptosis, will characterize an entirely novel interaction between components of the retromer complex, VPS35 and VPS26, and components of the BCL-2 family, BCL-xL. To answer fundamental questions about the relationship between proteins in two pathways that were considered distinct until now, we will use CRISPR/Cas9 gene-edited cells, confocal and single molecule super-resolution microscopy, co- immunoprecipitations and apoptosis assays. Overall, this proposal will make a unique contribution to cancer research by addressing the underlying mechanisms and upstream events that lead to the dysfunction of mitochondrial fission or mitochondrial induced apoptosis, which are severely impaired in cancer cells, and can be used as novel targets for cancer therapy.